Adverse proton electrochemical gradients (delta approximately mu H) applied across the turtle urinary bladder decrease active H ion transport in this epithelium. A delta approximately Mu H of 180mV abolishes both transport and its tightly coupled metabolic reaction . Larger gradients should, in theory, reverse the direction of H ion transport and the metabolic reaction leading to i synthesis of ATP if the pump is an ATPase, or cause an increase in the oxidized state of a redox pair if it is a redox pump. To distinguish between these two possibilities, we measured ATP levels in epithelial cells which were poisoned to inhibit cellular mechanisms of ATP synthesis. At delta approximately mu H of 120 mV or less no ATP synthesis was found. At delta approximately mu H of greater than 120mV there was a linear increase in ATP synthesis. Dinitrophenol (DNP), a H ion carrier, prevented synthesis at delta approximately mu H of 310mV. Dicyclohexylcarbodiimide (DCCD), an inhibitor of H ion transport that works at the cell surface, prevented ATP synthesis at delta approximately mu H of 310mV. These results demonstrate that a reversible proton-translocating ATPase in the mucosal border of the bladder is the H ion pump responsible for urinary acidification.